Human tuberculosis caused by Mycobacterium tuberculosis (M. tuberculosis) is a severe global health problem, responsible for approx. 3 million deaths annually, according to the WHO. The worldwide incidence of new tuberculosis (TB) cases had been falling during the 1960s and 1970s but during recent decades this trend has markedly changed in part due to the advent of AIDS and the appearance of multidrug resistant strains of M. tuberculosis. 
Organisms of the tuberculosis complex can cause a variety of diseases, but the commonest route of invasion is by inhalation of bacteria. This initiates an infection in the lung, which can ultimately spread to other parts of the body. Normally, this infection is restricted in growth by the immune system, so that the majority of infected individuals show few signs apart from cough and fever, which eventually abates. Approximately 30% of individuals are unable to contain the infection and they will develop primary disease, which in many cases will eventually prove fatal. However, it is believed that even those individuals who apparently control the infection remain infected, probably for the rest of their life. Certainly, individuals who have been healthy for years or even decades can suddenly develop tuberculosis, which has proven to be caused by the same organism they were infected with many years previously. M. tuberculosis and other organisms of the TB complex are unique in that the mycobacteria can evade the immune response and survive for long periods in a refractory non-replicating or slowly-replicating stage. This is referred to as latent TB and is at present a very significant global health problem which is estimated to affect approximately ⅓ of the world's population (Anon., 2001).
The only vaccine presently available for clinical use is BCG, a vaccine whose efficacy remains a matter of controversy. Although BCG consistently performs well in animal models of primary infection, it has clearly failed to control the TB epidemic. Consistent with that, BCG vaccination appears to provide protection against pediatric TB (which is due to primary infection), while offering little or no protection against adult disease (which is often reactivation of latent infection acquired in childhood). It has also been shown that vaccination of individuals who are currently sensitized to mycobacteria or latently infected is ineffective.
The course of a M. tuberculosis infection runs essentially through 3 phases, as illustrated in FIG. 1. During the acute phase, the bacteria proliferate in the organs, until the immune response increases to the point at which it can control the infection whereupon the bacterial load peaks and starts declining. After this, a latent phase is established where the bacterial load is kept stable at a low level. In this phase it has been the current thinking that M. tuberculosis goes from active multiplication to a state of dormancy, essentially becoming non-replicating and remaining inside the granuloma.
However, recently it has become clear that even in the stage of infection characterized by constant low bacterial numbers at least part of the bacterial population remain in a state of active metabolism (Talaat A M et al. 2007). These bacteria therefore survive, maintain an active metabolism and replicate in the face of a strong immune response. In the infected individual there is therefore a balance between non-replicating bacteria (that may be very difficult for the immune system to detect as they are located intracellularly) and slowly replicating bacteria that has an active but changed expression profile in an attempt to adapt to the hostile environment encountered in the immune host. Bacteria in this stage are typically not targeted by most of the preventive vaccines that are currently under development in the TB field as exemplified by the lack of activity when classical preventive vaccines are given to latently infected experimental animals (Turner 2000).
In some cases, the balance is tilted in favor of the pathogen and the infection goes into the reactivation phase, where the bacteria start replicating rapidly and bacterial numbers in the infected individual increases. Bacteria that replicates in latently infected individuals under very strong immune pressure is the target for the vaccination strategy in the present invention. Bacteria in this latent infective stage are typically not targeted by most of the preventive vaccines that are currently under development in the TB field as exemplified by the lack of activity when preventive vaccines are given to latently infected experimental animals (Turner et al 2000). This is not surprising as it is now known that a strong host immune response results in the down regulation of many antigens such as the major preventive vaccine antigen Ag85 and PstS (Rogerson, B J et al 2006). For Ag85B it has been shown that after infection there is an initial transient increase in Ag85B expression but already after two weeks infection the level of bacterial Ag85B expression had dropped from 0.3 transcripts per CFU of M. tb during the peak period to 0.02 transcripts per CFU and this low level is maintained at least up to 100 days post infection. Thus at any time point after week 2 of infection less than 2% of the bacteria actively express Ag85B (ibid.). The low expression of Ag85B is supported by a rapid drop in the number of T cells capable of making IFN-γ in response to Ag85B in the lung 3 weeks post infection or later.
In contrast some antigens are more stably (constitutively) expressed throughout the different stages of infection and one example of this is ESAT6. After the initial infection phase the ESAT-6 expression level stabilizes at 0.8 transcripts per CFU M. tuberculosis. This is a transcription level much higher than for Ag85B and this level is maintained stably up to at least 100 days post infection (Rogerson, B J et al 2006). Again transcription data is supported by immune data that shows strong T cell recognition of ESAT-6 at the later stages of infection at the site of infection (ibid.). This constitutive expression pattern is an important feature that illustrates that these molecules fulfills essential functions of crucial importance for the pathogen, functions that depends upon genes that needs to be constitutively expressed for the pathogen to survive in the immune host. These molecules are the basis for the current invention and are particularly important antigens for vaccines administered to latently infected individuals as they targets all stages of the bacterial lifestyle and therefore has the broadest possible basis for activity. This is different from current thinking that has been focused on identifying the antigens upregulated by mycobacteria during non-replicating persistence (Andersen, P. 2007, WO02048391, WO04006952, Lin M Y and Ottenhoff T H 2008; Leyten E M. et al. 2006). Although such antigens are upregulated during non-replicating persistence they may not always be available for immune recognition as the amounts available from non-replicating bacteria are below a reasonable threshold for detection or for the triggering of protective immune effector functions.
In contrast, several of the proteins from the ESX-1 secretion system have been shown to be highly immunogenic and expressed at high levels. ESX-1 is conserved in several pathogenic mycobacteria and involved in virulence of tubercle bacilli. The contribution of the individual ESX-1 proteins in secretion of ESAT-6, CFP10 and EspA has been well documented (Pym A S et al 2003; Guinn K I et al, 2004; Stanley, S A et al. 2003; Brodin, P. et al. 2006; MacGurn J A et al. 2005; Raghavan, S. et al. 2008) and the function of the effector molecules has been shown to be membrane lysis, escape from the phagosome and bacterial spreading (Gao L Y et al 2004; Smith J. et al. 2008).
The full nature of the immune response that controls latent infection and the factors that lead to reactivation are largely unknown. However, there is some evidence for a shift in the dominant cell types responsible. While CD4 T cells are essential and sufficient for control of infection during the acute phase, studies suggest that CD8 T cell responses are more important in the latent phase (van Pinxteren L A et al. 2000).
As one skilled in the art will readily appreciate, expression of a gene is not sufficient to make it a good vaccine candidate. The only way to determine if a protein is recognized by the immune system during latent infection with M. tuberculosis is to produce the given protein and test it in an appropriate assay as described herein. In this regard, our group has demonstrated that antigens strongly expressed by mycobacteria, such as ESAT-6 (Early Secretory Antigen Target-6) are recognized in individuals in all stages of infection and in fact in particular in latently infected individuals (Ravn 1999, Doherty 2002). However the ESAT-6 specific T cells primed during the natural infection are although they may be present in large numbers, almost exclusively of the so-called effector phenotype that are terminally differentiated T cells with a very limited lifespan and of low activity as protective T cells against infectious diseases (Seder R, et al. 2008). This is markedly different from the high quality, so-called polyfunctional T cells that are promoted by the vaccine demonstrated in the present study to protect against reactivation of TB.
It is far from all highly expressed and immunogenic proteins that are useful as post exposure vaccines because many will provoke hypersensitivity reactions and thereby worsen the situation instead. This was clearly demonstrated in the clinical trial of Koch's original tuberculin vaccine. The vaccine was given as a post exposure vaccine to patients suffering from different forms of the disease including skin and pulmonary TB. The trial was a complete failure and several of the enrolled patients died because of severe hypersensitive reactions (Guttstadt A. 1891). Of the several hundred antigens known to be expressed during primary infection, and tested as vaccines, less than a half dozen have demonstrated significant potential. So far only one antigen has been shown to have any potential as a post-exposure vaccine (Lowrie, 1999). However this vaccine only worked if given as a DNA vaccine, an experimental technique so far not approved for use in humans. Moreover, the technique has proved controversial, with other groups claiming that vaccination using this protocol induces either non-specific protection or even worsens disease (Turner, 2000).
Therefore, an effective postexposure vaccination strategy to protect infected individuals against reactivation of the disease is highly desirable.